The major questions we focus on are: 1) What are the upstream regulatory components that specify the animal pole domain (APD) of the sea urchin embryo, which contains nerves and cells bearing long, immotile cilia? 2) What are the signals transmitted during early cleavage and blastula stages that are required for endomesoderm specification and timely gastrulation? 3) What is the molecular basis for the embryo's ability to change its morphology to adapt to changing food concentrations? In each case, we assay the expression of all predicted genes in the genome in order to gain a complete understanding of the gene regulatory networks that underlie these processes. Six3 is necessary and sufficient for APD specification (50%) (Zheng Wei, Ryan Range, Lynne Angerer) During the last several years, using bioinformatics and molecular screens, we identified many genes encoding regulatory proteins expressed specifically in the primary neurogenic domain of the sea urchin embryo. Two of the earliest, FoxQ2 and Six3, control early decisions in ectodermal patterning. Experiments conducted during 2008 showed that Six3 is necessary and sufficient for all known features of APD development. This includes all neurons formed during embryogenesis and the large majority of APD-specific regulatory proteins previously identified, including FoxQ2. We identified the full regulatory repertoire of genes that depend on Six3, and discovered that this factor can also suppress canonical Wnt and TGF-beta signals that pattern the rest of the embryo. Our work revealed that the function of Six3 in the sea urchin embryo APD shares some features with Six3 in the vertebrate forebrain. Our studies have identified many additional regulatory genes that are Six3-dependent and expressed in the APD. It is therefore of interest to determine whether the vertebrate orthologs of these genes function in vertebrate forebrain development. This work has been published by Wei et al., Development 136, 1179-1189 (2009). FoxQ2 not only can suppress TGF-beta signaling in the APD(Yaguchi et al., Developmental Cell 14, 97-107, 2008), but it also required for controlling Wnt signaling in this territory. Microarray screens show that it is required for the expression of nearly 500 genes including a subset of the genes encoding early APD regulatory proteins and for specialized cilia specifically in the APD. Further, FoxQ2 is required for production of regulators of Wnt signaling. When these regulators are either eliminated or over expressed, the position of the boundary of the APD within the ectoderm is altered. These results reinforce the model that mutual antagonism between the outputs of the APD GRN and Wnt signaling regulates APD development within the early ectoderm of the sea urchin embryo. Activation of the APD GRN depends upon maternal positive regulatory activities. One of these is SoxB1. When SoxB1 is eliminated, differentiation of nerves within the APD is blocked and expression of the key upstream components of the APD GRN, Six3, FoxQ2 and Hbn (homeobrain) are reduced in the blastula-stage embryo. SoxB1 also plays a key role in activating the oral and aboral ectodermal developmental GRNs as well as in suppressing canonical Wnt signaling. Determining how the relative contributions of SoxB1 to ectodermal patterning are controlled will be critical for understanding the initial events that specify cell fates within the ectoderm. ActivinB/ALK4-5-7 and Delta/Notch are early signals that induce specification of endomesoderm and endoderm (25%)(Adi Sethi, Radhika Wikramanayake, Lynne Angerer) Previous experiments showed that both ectopic and normal endomesoderm induction requires ActivinB and that this signaling factor executes all of the endomesoderm inducing functions of the previously unknown, but long-sought, early micromere signal. This work (Sethi, AJ, Angerer, RC, Angerer, LM (PLOS Biology 7(2): ee1000029, 2009) is the first to connect ActivinB signaling to specific components of an endomesoderm gene regulatory (GRN) network, the largest and best developed in any embryo, and it has led to new insights into its operation. Experiments in which Delta/Notch signaling is blocked reveal new functions for this pathway in separating two primary germ layers, the endoderm and mesoderm, within the endomesodermal field. Micromere Delta signals not only activate gcm in overlying macromere progeny, but also repress several critical genes required for early endoderm specification. Recent results suggest that a key element in the specification of presumptive mesodermal cells is the ability of Notch signals to down regulate the canonical Wnt signaling pathway. Dopaminergic neurons regulate the embryo's response to food density (25%) (Diane Adams, Lynne Angerer) A new line of investigation aims to understand the molecular pathways by which larvae sense and respond to food concentration by adjusting arm length to maximize food intake. It is a high-risk project that bridges larval developmental biology, larval physiology and the ecology of larval dispersal. Diane has found that dopamine D2receptor activity regulates changes in the post-oral skeletal rod length in response to food density. She has identified cells expressing dopamine and tyrosine hydroxylase in the post-oral arms where the response to food density occurs. Experiments are underway to identify which D2 receptor mediates this response. To determine other components of the food sensing pathway, she is using microarrays to assay the whole genome reponse to food concentration as well as to dopamine antagonists and agonists.